Classically, vaccines against enveloped viruses either contain killed or live attenuated viruses, or they comprise a preparation of their constituents (e.g. split virus or subunit preparations). For vaccination, these preparations are usually injected. After injection, the viruses or proteins present in such vaccines are taken up by antigen-presenting cells of the immune system such as dendritic cells or macrophages, followed by a presentation of the antigenic parts of the vaccines to effector cells of the immune system. Vaccines are effective when injected because antigen-presenting cells are most abundant just under the skin. However, it has now become clear that similar cells are also present in the mucosa that, for instance, lines the nose (Ogra et al. 2001). In order to induce these phagocytes present in the mucosa to mount an immune response, much stronger stimulation is required than for those present under the skin (Janeway et al. 2001).
While the injection of some viruses or proteins contained in vaccines, for example influenza or measles virus, elicits an immune response that is sufficiently strong to protect against a later infection by the same virus, this is not the case for many others, for example respiratory syncytial virus. Numerous attempts to reinforce the immune response by physical or chemical means have been undertaken. The most important principles that have emerged from such experiments are: (1) for physical stimulation, multiple copies of the viral proteins need to be combined in particles. These particles can be whole viruses, reconstituted viral membranes, or proteins on microparticle carriers. Particles stimulate the immune system better than individual subunits (Ogra et al. 2001; Janeway et al. 2001). (2) Chemical stimulation on the other hand requires that the phagocytes or the effector cells of the immune system receive certain signals through receptors present on surface of the antigen-presenting cell, for instance through the use of adjuvants, chemical compounds that are recognized by these receptors.
With sufficient additional physicochemical stimulation, viral proteins can elicit strong immune responses even if applied to mucous membranes, for example upon application to the nose (Ogra et al. 2001). Most of the current methods and compositions for stimulating an immune response by such means, whether by chemical or physical means or combinations of the two principles, have significant disadvantages that will be outlined below.
A particular kind of vaccine composition that was developed in the art is known as ‘virosomes’, which are lipid bilayers containing viral glycoproteins. Virosomes may comprise reconstituted viral membranes, generally produced by extraction of membrane proteins and lipids from enveloped viruses with a detergent, followed by addition of lipids, and removal of said detergent from the extracted viral membrane proteins and lipids, such that characteristic lipid bilayers are formed with the proteins protruding from them (Stegmann et al. 1987). Virosomes may also comprise membranes formed from purified viral proteins and synthetic or natural lipids, or other substances that will form a bilayer. A characteristic feature of virosomes is that they closely mimic the composition, surface architecture and functional activities of the native viral envelope. A particularly important characteristic of said virosomes involves the preservation of the receptor-binding and membrane fusion activity of the native viral envelope, allowing the virosomes to enter the same cells that the virus would be able to enter, and to be presented to the immune system by these same cells. Preservation of receptor-binding and membrane fusion activity is essential for expression of the full immunogenic properties of said virosomes (Arkema 2000; Bungener 2002).
For some viral antigens, virosomes elicit protective immune responses that can be strong even when the vaccine is, for example, delivered intranasally (as is exemplified in WO 88/08718 and WO 92/19267). However, other virosome formulations exhibit only marginally improved immunogenicity as compared to killed virus or subunit preparations (as exemplified in (Glück et al. 1994). In this cited example, the virosomes were generated through a protocol involving addition of exogenous lipids, which we have found to result in a composition of the virosomes and a surface architecture different from those in the native viral envelope. It is known to a person skilled in the art that such a different surface architecture may affect the membrane fusion properties of the virosomes produced and thus their immunogenicity.
To enhance the immune response, allowing intranasal application of this vaccine, an adjuvant protein from Escherichia coli (heat-labile toxin) was mixed with the lipid-supplemented virosome influenza vaccine (EP 0 538 437). Clinical trials indicated that addition of the toxin was absolutely required to induce serum antibody titers equivalent to injected vaccine (Glück et al. 1994). Although addition of the toxin did thus enhance the immunogenicity of this vaccine, it also induced a serious side effect known as Bell's Palsy, a temporary paralysis of facial muscles. Since the adjuvating effect of the toxin is due to recognition by an antigen-presenting cell, there is no certainty in this case that the toxin and the viral protein will contact the same cell, and therefore a relatively high concentration of the toxin will be needed in order to ensure activation of every cell, increasing the chance that antigens will be recognized by an activated cell. Therefore this type of virosome preparation with added lipids has a fair number of disadvantages.
Virosomes have also been prepared from purified influenza antigens, mixed with derivatives of muramyldipeptide (EP 0 205 098 and EP 0 487 909). In this case, the muramyldipeptide derivative forms the membrane. Although muramyldipeptide is an adjuvant, and the formulation was indeed found to enhance the immune response to the influenza antigens, muramyl dipeptides are pyrogenic (Kotani et al., 1976; Dinarello et al., 1978), are cleared rapidly from the body following injection, and have local toxicity leading to granulomas and inflammation (Ribi et al., 1979; Kohashi et al., 1980). Moreover, they have a limited shelf life at neutral pH (Powell et al., 1988), and the optimal pH to maintain their structural integrity is too low to allow their formulation in a vaccine together with the fusion protein of viruses that enter cells by receptor-mediated endocytosis, such as the hemagglutinin of influenza virus. Moreover, such synthetic membranes are not a good mimic of the natural viral membrane and thus the immune response to them will differ from that generated against the virus.
Alternatively, researchers in the art have also generated complexed antigens different from reconstituted viral membranes, such as ‘Immunostimulatory Complexes’ (ISCOMs, Morein et al. 1984), containing viral proteins complexed with adjuvants such as saponins like Quil A® (EP 0231039B1; EP 0109942A1; EP 0180564A1), most of which are isolated from the bark of Quillaia sopanaria Molina. Mixed with antigen, and lipids such as cholesterol, these adjuvants form cage-like structures of between 30-40 nm, rendering the antigen particulate, while acting at the same time as an adjuvant. Although ISCOMs have been used in a number of veterinary vaccines, and enhance the immunogenicity of the viral membrane proteins, the development of such vaccines for humans has been inhibited by concerns about their toxicity and the complexity of the mixture (Cox et al. 1998).
More recently, proteosome influenza vaccines were developed (US application 20010053368), consisting of non-covalent complexes of the purified outer membrane proteins of bacteria such as meningococci, mixed with antigenic proteins such as the influenza hemagglutinin or the human immunodeficiency envelope glycoprotein. While these multiple bacterial proteins may act as adjuvants, the complex nature of such mixtures, consisting of multiple proteins, will present a regulatory issue. Moreover, the immune response is directed against all of the proteins and other antigens present in the solution, and less specifically against the viral proteins.
Another particulate formulation developed by Biovector Therapeutics consists of an inner core of carbohydrate surrounded by a lipid envelope containing antigens. With influenza hemagglutinin as the antigen, some enhancement of the immune response was noted, but not significant enough to warrant further development.
Live attenuated versions of respiratory viruses, such as a cold-adapted strain of influenza virus with minimal replication in the respiratory tract have been developed as intranasal vaccines. These vaccines have the distinct advantage of inducing immune responses that are close to the natural immunity induced by an infection with wild-type virus. For influenza, such vaccines have been known since the 1980's, and now appear close to commercialization. The delay has been caused by the ability, that many viruses share, to mutate rapidly, causing the attenuated viruses to revert partially of wholly to wild-type virus, and thereby in fact causing the disease they were meant to prevent.
For the above reasons, it is well recognized in the art that, especially to induce immune responses for pathogens that do not by themselves induce a strong immune response, and for intranasal and other mucosal applications, although compositions such as ISCOM's and proteosomes were developed, there still is a great need for well characterized vaccine compositions that induce a strong immune response, do not contain live virus, and have a low toxicity.